Minimal Transgenerational Effect of ZnO Nanomaterials on the

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Minimal transgenerational effect of ZnO nanomaterials on the physiology and nutrient profile of Phaseolus vulgaris Illya A Medina-Velo, Nubia Zuverza-Mena, Carlos Tamez, Yuqing Ye, Jose A. HernandezViezcas, Jason C. White, Jose R Peralta-Videa, and Jorge L. Gardea-Torresdey ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01188 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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ACS Sustainable Chemistry & Engineering

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Minimal transgenerational effect of ZnO nanomaterials on the physiology and nutrient

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profile of Phaseolus vulgaris

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Illya A. Medina-Velo1,4, Nubia Zuverza-Mena2, Carlos Tamez3, Yuqing Ye1, Jose A. Hernandez-

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Viezcas1,4, Jason C. White2, Jose R. Peralta-Videa1,3,4 and Jorge L. Gardea-Torresdey1,3,4,* 1

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Department of Chemistry, The University of Texas at El Paso; 500 West University Ave., El

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Paso, TX 79968, United States. 2

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Huntington St., New Haven, CT 06504, United States 3

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Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, 123

Environmental Science and Engineering Ph.D. Program, The University of Texas at El Paso; 500 West University Ave., El Paso, TX 79968, United States.

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University of California Center for Environmental Implications of Nanotechnology (UC CEIN),

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The University of Texas at El Paso; 500 West University Ave., El Paso, TX 79968, United

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States.

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Corresponding author: [email protected] (J. Gardea); Phone: 915-747-5359; Fax: 915-747-5748.

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ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Bean (Phaseolus vulgaris) plants were cultivated in nanoparticle-unamended soil and their

3

second-generation seeds (S2) were analyzed to determine the transgenerational effects of

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uncoated nano-ZnO (Z-COTE), coated nano-ZnO (Z-COTE HP1), bulk ZnO, or ionic Zn

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(ZnCl2) at 0-500 mg kg- 1. Antioxidant enzymatic activity was determined in immature seeds,

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while the yield and nutritional composition of the seeds were measured at the end of the growth

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cycle. None of treatments affected seed production, maturation time, Zn accumulation, or the

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content of sugar, starch or protein in S2, compared with controls. The accumulation of K, P, S,

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Mg, Fe, Mn, B, Mo, and Cu in S2 seeds also remained unaffected. However, Z-COTE at 500 mg

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kg-1 and Z-COTE HP1 at 125 and 500 mg kg-1 reduced Ni in S2 seeds by 60%, 41%, and 74%,

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respectively, compared with the control. The Zn nanomaterials (NMs) did not impact the activity

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of ascorbate peroxidase, catalase, and superoxide dismutase (SOD) in the seeds. However, ZnCl2

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at 500 mg kg-1 increased SOD by 28% in S2 seeds. The data suggest that ZnO NMs have low

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residual transgenerational effects on seed composition, which could be beneficial in agricultural

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production.

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Keywords: Z-COTE, transgenerational, ZnO nanoparticles, nutrients, production, coated, beans,

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plants

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Introduction

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The fate of nanomaterials (NMs) incorporated into personal care products (PCPs) is still largely

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unknown. It has been estimated that 28-32% of such NMs are released to bodies of water.1

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Particles such as ZnO NMs, Z-COTE, and Z-COTE HP1 are widely used in PCPs. The bare ZnO

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NM (Z-COTE) has an amphiphilic nature and is used in water-based formulations; Z-COTE

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HP1, has a hydrophobic coating of triethoxycaprylylsilane and is suitable for oil-based

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formulations.

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ZnO NMs can reach agricultural environments unintentionally when released from PCPs or

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intentionally as agrochemicals.5 These NMs, and the released ions, can also reach agricultural

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fields through irrigation with NM-containing water or biosolids.2-4 The effects of ZnO NMs on

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plants and the accumulation of Zn after exposure has been studied.6,7 Physiological and

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biochemical effects of such nanomaterials in exposed wheat,2 soybean,3,4 peanut,5 kidney bean,6

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cucumber,7 pea,8 and sorghum have been reported.9 However, the residual effects of the

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exposure to ZnO NMs across multiple plant generations (MG) remains unexplored.10 Long-term

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exposure to NMs may cause stress and possibly physiological adaptations.11 The adaptation

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process can be associated with phenotypic or genotypic changes,12 or by epigenetic

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modifications (i.e., changes in gene function that cannot be explained by alterations in the DNA

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sequence).13 Several contaminants have been reported to produce epigenetic changes in plants

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such as Arabidopsis thaliana upon exposure to ZnO, TiO2, and fullerene soot,19,14 and in Zea

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mays in response to Zn stress.15 Furthermore, transgenerational epigenetic effects can be

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observed in non-exposed plants from the subsequent generation if the effect on the maternal

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gene function prevails.12

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1

The assessment of plant response to NMs commonly includes determining the impact on

2

biomass, growth, yield, bioaccumulation of NM/ions, and nutrient composition. In addition,

3

given that NMs can interact with biological molecules, stress biomarkers such as increases in

4

reactive oxygen species (ROS) can be used as an assessment of toxicity.16,13 Plants respond to

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excess ROS through antioxidant defense systems by converting ROS into less damaging

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species.17,18 Antioxidant enzymes include superoxide dismutase (SOD), which catalyzes the

7

dismutation of superoxide radical (O2.-) to H2O2 and O2,19,20 ascorbate peroxidase (APX), and

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catalase (CAT); the latter two convert H2O2 to H2O.

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Only a few intergenerational studies on the effects of NMs in plants are found in the literature. In

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Brassica rapa, the consecutive exposure of three generations to CeO2 NMs showed higher

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oxidative stress and lower seed production in the later generations.21 Rico et al.22 reported

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changes in the physiology and nutrient profile of the second generation of wheat (Triticum

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aestivum) plants exposed to CeO2 NMs. The MG exposure to CeO2 produced grains with lower

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Mn, Ca, K, Mg, and P, compared with single exposure.22 However, to the authors’ knowledge,

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studies on the residual effects of the exposure to Z-COTE and Z-COTE HP1 have not been

16

reported. Hence, the objective of this study was to measure the nutrient profile and

17

physiological status in second generation bean seeds (S2). The seeds from the first generation

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(S1) were obtained from plants cultivated in soil amended either with Z-COTE, Z-COTE HP1,

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bulk ZnO, or ZnCl2 at 0-500 mg/kg.6 Then, S1 seeds were grown in clean soil and S2 were

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analyzed. Spectroscopic and biochemical techniques were used to evaluate the residual effects of

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the exposure to Zn nanomaterials or compounds by measurement of Zn accumulation, nutritional

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profiles, and antioxidant activity in the S2 seeds.

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Experimental Section

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Nanomaterials, compounds, and soil

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Bulk ZnO (≥ 1000 nm, ACS reagent ≥ 99% purity) was purchased from Sigma Aldrich; Z-

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COTE [average diameter= 93.8 nm; size in deionized water (DW, 50 µg mL-1)= 286 ± 2 nm], Z-

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COTE HP1 (average diameter= 84.1 nm; size in DW= 276 ± 7 nm)23 were purchased from

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BASF; and ionic ZnCl2 (ACS reagent ≥ 97% purity) was purchased from Acros Organics.

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Nanomaterial characterization has been previously reported.23 Maternal bean seeds (Phaseolus

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vulgaris var red hawk) (S0) were supplied by Dr. James Kelly from Michigan State University

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and stored at 4°C until experimentation.24 Medium loam soil was collected from an agricultural

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field in Texas (N 31° 40.489′, W 106° 17.198′, elevation: 1115 masl) and commercial Miracle

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Grow® potting mix was purchased from a local store.25 Both soils were sieved through an 8 mm

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mesh to exclude larger materials and a mixture for plant growth was prepared by mixing 250 g

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of medium loam soil and 250 g of potting mix (50:50 %wt).

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Maternal seed exposure and growth

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Four replicates of bulk ZnO, Z-COTE, Z-COTE HP1, and ZnCl2 (powders) were weighed to

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achieve concentrations of 125, 250 and 500 mg kg-1 soil (compound based) and were suspended

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in 50 mL of DW, as previously described.26 Bulk ZnO and Z-COTE suspensions were sonicated

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in a water bath (Crest Ultrasonics, Trenton, NJ) for 30 min at 25 ◦C and 180 watts. Without

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sonication, ZnCl2 was dissolved and Z-COTE HP1 was manually mixed into solution. The

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suspensions/solutions were added to the soil, mixed with a hand cultivator to optimize

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homogeneity, transferred into plastic pots (12.5 cm diameter × 14 cm height) and left at room

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temperature for 24 h. Four replicates of soil watered with DW were used as controls for each 5



1

compound. The S0 seeds were washed with 2% NaClO, rinsed three times with DW for

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disinfecting purposes, and soaked in DW to facilitate germination. After 24 h, five S0 seeds

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were planted in each pot at 2.5 cm depth and were watered with 50 mL of DW. The pots were

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transferred to a growth chamber (Environmental Growth Chamber, Chagrin Falls, OH) with 14 h

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photoperiod (340 µ mole m−2 s−1), 25/20 ºC day/night temperature, and 65-70% relative

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humidity. The soil was watered daily with 50 mL of DW until germination, then with 100 mL

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DW (daily) until S1 seeds were mature (pods are yellow, dry, and start to open, and seed are red

8

with ~15% humidity).6 Although the experiment was set in a growth chamber with uniform

9

conditions, the pots were randomly rotated every 14 days to reduce any possible differences in

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illumination. At maturity (87 ± 11 days),6 pods were cut from the calyx, including the pedicel;

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the S1 seeds were removed from the pods, placed in plastic bags, and stored at 4°C.

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Growth of second generation plants

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The S1 seeds were disinfected with 2% NaClO, rinsed 3 times with DW, and soaked in DW for

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24 h. Three seeds from each treatment/replicate were planted at 2.5 cm depth in plastic pots

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containing 500 g of uncontaminated soil and watered with 50 mL DW. The pots were transferred

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to an environmental growth chamber with the same conditions used for the first generation. All

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seeded pots were watered with 50 mL DW until germination, then with 100 mL until maturity,

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and were randomly rotated every 14 days. Forty-five days after planting, when all pods were

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fully developed, one pod from each plant/replicate was collected and immature S2 seeds were

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obtained for enzyme extractions. Plants were then allowed to grow until maturity; pods were

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then collected, and the weight and number of S2 seed-containing pods was recorded. The S2

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seeds were removed from the pods and the total number of seeds was recorded, along with the

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weight of the pods and seeds. The S2 seeds were placed in paper envelopes and oven dried at 70

2

°C for 72 h.

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Antioxidant enzymatic activity in young seeds

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The APX (EC 1.11.1.11) activity was determined according to Bailly et al. (2001)26 and Nakano

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and Asada (1981),27 and CAT (EC 1.11.1.6) as reported by Bailly (1996).28 The SOD (EC

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1.15.11) activity was estimated by measuring inhibition of the photochemical reduction of

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nitroblue tetrazolium (NBT) by the enzyme extract.19,28–31 The detailed methods are summarized

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in the Supporting Information.

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Quantification of total sugar, starch, and protein

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Sugar and starch were extracted from 0.1 g of dry seed as described by Verma and Dubey.32

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Total sugar and starch were quantified by the phenol-sulfuric acid method in a microplate

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format33 using a Spectra Max 190 Microplate Reader (Molecular Devices, San Jose, CA). The

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protein content was analyzed in a LECO FP628 Nitrogen Determinator (Saint Joseph, MI)

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following the nitrogen combustion method described by Chang.34 LECO reference materials of

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wheat flour and EDTA were used for quality control and quality assurance (QA/QC) during

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protein determination.

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Essential element quantification via ICP-OES

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Quantification of major (Ca, K, Mg, P, S) and trace (B, Cu, Fe, Mo, Mn, Ni, and Zn) minerals in

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S2 seeds was done with dry powdered samples. Samples of 0.1-0.2 g were pre-digested with 3

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mL of 70% HNO3 for 45 min at room temperature. For digestion, the samples were heated on a

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hot block digestion system (SCP Science, Champlain, NY) at 115 °C for 45 min followed by 7



1

dilution to 50 mL with ultra-pure water (UPW, 18.2 MΩ cm-1). The elemental composition was

2

measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES, iCAP 6500,

3

Thermo Fisher Scientific, Waltham, MA). The ICP-OES parameters were as follows: nebulizer

4

flow, 0.50 L/min; power, 1,150 W; peristaltic pump rate, 45 rpm; and flush time, 45 s. For QC of

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the ICP-OES readings, a multi-elemental standard solution of 1 mg/L was analyzed every 12

6

samples and yttrium was used as internal standard. The National Institute of Standards and

7

Technology (NIST) standard reference material 1570a (spinach leaves) was used to validate the

8

digestion and analytical method; a Zn analyte recovery of 93% was achieved.

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Statistical analysis

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The Statistical Package for the Social Sciences 22 (SPSS, Chicago, IL, USA) was utilized for

11

one-way ANOVA tests to evaluate the experimental variance. Differences between treatments

12

were evaluated with the multi comparison Tukey’s HSD test at a p-value of 0.05. Data presented

13

are means of specified number of replicates.

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Results and Discussion

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Days to reach maturity, number of plants, and yield

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Table 1 shows the data for number of days to reach maturity, number of developed plants, and

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pod/seed production of the second generation bean plants. None of the treatments affected these

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parameters relative to the control. Plants from the second generation matured at 107 ± 5 days,

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while plants from the first generation lasted 87 ± 11. Additionally, all S1 seeds pre-exposed to Z-

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COTE HP1 successfully germinated (= 3.0 ± 0.0).

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There was no transgenerational effect of treatments in the number and weight of seeds (Table 1),

2

However, the number of pods in the second generation was reduced (45%, p ≤ 0.05) by ZnCl2 at

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250 mg kg-1, compared with bulk ZnO at the same concentration. The treatments did not affect

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the S2 seed weight; however, the S1 seed weight from our previous study was decreased by 74%

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under exposure to ZnCl2 at 250 mg kg-1, compared with bulk ZnO at the same concentration.6 A

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negative effect in production caused by ZnCl2 has been attributed to the formation of choline

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chloride,35 formed by Cl- ions released from ZnCl2, or to a toxic effect of Cl- anion that has been

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reported to decrease plant growth and yield. 36,37 Overall, the results suggest minimal

9

physiological impact on the plants from parental pre-exposure to the nanomaterials; the effects

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of ZnCl2 were similarly inconsequential at genetic level since no impact was evident in the

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second generation of plants.

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Table 1. Time to reach maturity, number of plants, pods, and seeds by the second generation of bean plants cultivated in nanoparticle-free soil. Maternal plants were exposed to bulk ZnO, ZCOTE, Z-COTE HP1, and ZnCl2 at 0 (control), 125, 250 and 500 mg kg-1. Values are means of 4 replicates for Zn compounds and 16 for controls. Letters represent statistically significant differences between means of the different compounds at the same concentration (p ≤ 0.05). The treatment concentrations refer to S1 plants. Concentration -1

Pods

Compound

(mg kg )

Days (#)

Plants (#)

***

Control

102.5

2.8

125

105.0

250

111.5

500

Bulk ZnO

Z-COTE

Z-COTE HP1

ZnCl2

Number (#)

Seeds

Weight (g)

Number (#)

Weight (g)

8.0

ab

12.8

20.8

2.3

7.8

ab

11.9

17.5

8.2

3.0

11.0

a

15.6

23.8

10.8

97.3

3.0

9.0

ab

13.1

21.3

8.6

125

110.5

2.8

8.8

ab

13.2

20.3

9.0

250

114.8

3.0

10.0

ab

13.9

22.3

9.6

500

108.3

2.5

8.3

ab

14.2

21.3

9.8

125

96.0

3.0

7.3

ab

11.0

16.8

7.2

250

109.8

3.0

8.5

ab

14.5

22.0

10.3

500

106.5

3.0

9.8

ab

15.8

23.3

10.6

125

107.8

2.0

7.3

ab

13.4

20.5

9.5

250

109.8

3.0

6.0

b

9.4

13.8

6.5

500

115.3

2.8

7.8

ab

14.0

22.0

9.6

9


8.9


1

2

Total sugar, starch and protein

3

Quantification of sugar, starch, and protein content in the S2 seeds is shown in Table 2. Similar

4

to the growth/production parameters, none of the treatments significantly affected sugar, starch,

5

and protein content, compared with the control, which had 4.8%, 40.8%, and 22.9%,

6

respectively. The values of protein and sugar are within the standard reference for red kidney

7

bean (sugar- 2.10% and protein- 22.53%) according to the USDA.38 These values are an

8

indicative of healthy seeds. However, parental exposure to ZnCl2 reduced S2 seed sugar content

9

by 27% at 500 mg kg-1, comparison to Z-COTE HP1 at the same concentration. In addition, in

10

S2 from the Z-COTE HP1 treatment at 500 mg kg-1, the starch content trended higher (49.5%)

11

than control (40.8%), although the difference was not statistically significant. Our previous study

12

showed that exposure to ZnCl2 at 125 and 250 mg kg-1 significantly increased the relative sugar

13

content (148% and 117%, respectively) in S1 compared with control seeds.6 Also in S1, the 500

14

mg kg-1 Z-COTE HP1 and ZnCl2 treatments resulted in similar amounts of sugar, and regardless

15

of concentration, the relative sugar content in seeds exposed to ZnCl2 was higher in comparison

16

to the rest of the compounds. The enhanced sugar accumulation in S1 was attributed to a plant

17

response to osmotic stress18 caused by the presence of Cl-;6 once the stress was removed, the

18

sugar concentrations returned to the normal levels.

19

Zinc accumulation

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In our previous study, all materials, at all concentrations, increased S1 Zn content in a dose-

21

dependent fashion, with values up to 147% higher than control seeds.6 However, the

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accumulation of Zn in S2 seeds was not significantly affected, compared to control (Figure 1).

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Interestingly, a comparison of the residual effects across compounds showed differences 10 ACS Paragon Plus Environment

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between bulk ZnO and ZnCl2. The S2 seeds harvested from S1 plants exposed to bulk ZnO at

2

500 mg kg-1 had 56% more Zn than seeds from plants exposed to ZnCl2 at the same

3

concentration. The reasons for this result are unknown. In S1 seeds, bulk ZnO and ZnCl2, at 500

4

mg kg-1, resulted in similar amount of Zn;6 thus, the Zn content in S2 cannot be due to a higher

5

reserve of Zn. An additional study is needed to understand this difference in bulk and ionic

6

exposure and effect.

7 8 9 10 11 12

Table 2. Sugar, starch, and protein content in second generation bean seeds cultivated from plants grown in nanoparticle-free soil. Maternal plants were exposed to bulk ZnO, Z-COTE, ZCOTE HP1, and ZnCl2 at 0 (control), 125, 250 and 500 mg kg-1. Values are means of 4 replicates for Zn compounds and 16 for controls. Letters represent statistically significant differences between means of the different compounds at the same concentration (p ≤ 0.05). The treatment concentrations refer to S1 plants. Compound

Concentration (mg kg-1)

***

Sugar (%)

Starch (%)

Protein (%)

Control

4.8

ab

40.8

22.9

125

5.3

ab

33.5

23.6

250

5.1

ab

56.6

23.2

500

4.3

ab

41.5

22.2

125

5.3

ab

47.4

22.8

250

4.4

ab

48.5

23.4

500

5.1

ab

45.7

21.4

125

4.5

ab

34.4

22.2

250

5.2

ab

46.8

23.0

Bulk ZnO

Z-COTE

Z-COTE HP1

ZnCl2

500

5.5

b

49.5

22.3

125

5.6

b

40.6

22.4

250

5.4

ab

50.6

21.6

500

4.0

a

35.9

21.0

13 Compound *** Bulk ZnO

Z-COTE Z-COTE

Concentratio n (mg kg-1) Control 125 250 500 125 250 500 125

Sugar (%) 4.796 ± 0.200ab 5.324 ± 0.393ab 5.138 ± 0.291ab

Starch (%) 40.83412 ± 4.025a 33.4812 ± 5.007a 56.56811 ± 5.021a

Protein (%) 22.920 ± 0.425a 23.643 ± 0.267a 23.232 ± 0.946a

4.285 5.321 4.422 5.108 4.537

41.48131 47.4108 48.5103 45.71988 34.36022

22.188 22.800 23.375 21.449 22.237

± ± ± ± ±

0.056ab 0.281ab 0.285ab 0.281ab 0.493ab

11


± ± ± ± ±

8.921a 10.180a 14.948a 6.845a 2.631a

± ± ± ± ±

0.601a 1.059a 0.965a 3.586a 1.439a


HP1

250 500 125 250 500

ZnCl2

5.201 5.541 5.615 5.429 3.965

± ± ± ± ±

0.194ab 0.110b 0.265b 0.427ab 0.146a

46.79126 49.46662 40.64136 50.60306 35.91912

± ± ± ± ±

6.588a 7.543a 2.659a 7.591a 1.737a

22.957 22.326 22.436 21.586 21.013

± ± ± ± ±

0.536a 1.055a 0.891a 1.039a 0.571a

1 2

The fact that ZnO NMs allows Zn accumulation in seeds, without transgenerational effects,

3

represents a viable scenario for their use in Zn-deficient soils.39 This represents a possibility to

4

fight Zn deficiency in humans.

5

Essential elements

6

In addition to Zn, major (Ca, Mg, K, P, S) and trace minerals (B, Cu, Fe, Mo, Mn, and Ni) were

7

analyzed in S2 seeds. All essential elements in S2 except Ca and Ni were unaffected by parental

8

S1 exposure (Figure 2). The accumulation of K, P, Mg, and Fe in S2 are within the normal

9

values, per the report of the USDA38 (K= 13,590.0 mg kg-1, P= 4,060.0 mg kg-1, Mg=1,380.0

10

mg kg-1, and Fe= 66.9 mg kg-1); unfortunately, the remaining elements listed on Figure 2 are

11

not reported by the USDA.

Control 40

Zn (mg · kg dry seeds-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 25

125

250

500

[Zn]

a

35

ab

30

ab

25

b

20 15 10 5 0

12 13 14 15 16

Bulk ZnO

Z-COTE

Z-COTE HP1

ZnCl2

Figure 1. Zinc content in second generation bean seeds cultivated from plants grown in nanoparticle-free soil. Maternal plants were exposed to bulk ZnO, Z-COTE, Z-COTE HP1, and ZnCl2 at 0 (control), 125, 250 and 500 mg kg-1. Values are mean ± SE of 4 replicates for Zn compounds and 14 for controls. Letters represent statistically significant differences between

12


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1 2


means of the different compounds at the same concentration (p ≤ 0.05). The treatment concentrations refer to S1 plants.

3 4

The accumulation of Ca and Ni was significantly affected in seeds of the second generation

5

(Figure 3). Parental exposure to bulk ZnO at 500 mg kg-1 increased Ca accumulation in S2 by

6

51% compared with the control seeds. In S1, bulk ZnO showed the highest amount of Ca (2,384

7

mg kg-1) across all treatments/concentrations.6 It is possible that such Ca levels in S1 caused

8

epigenetic effects that promoted the formation of more Ca2+-permeable cation channels, which

9

are responsible for Ca delivery to the plant tissues.12,40

10

Alternatively, both ZnO nanoparticles reduced Ni in S2. Z-COTE at 500 mg kg-1 reduced S2 Ni

11

by 60%, while Z-COTE HP1, at 125 and 500 mg kg-1, reduced Ni by 41% and 74%, respectively,

12

compared with control seeds. Similar reductions in Ni uptake by ZnO NMs exposure were

13

reported in S1 seeds at exposures of 125, 250, and 500 mg kg-1. This finding was found to be

14

nanoscale-specific,6 since the Ni reductions were observed only with NM exposure and not the

15

conventional microsized compounds. It has been reported that Ni2+ and Zn2+ are transported by

16

the same carrier protein system,41 which explains the Ni reductions in S1 seeds and suggests the

17

influence of the Zn particle size in the mechanisms of Ni absorption. Importantly, the residual

18

effect of ZnO NMs on S2 seeds suggests genetic or epigenetic changes in the transport

19

mechanisms of Ni. Zinc accumulation produces hypomethylation of certain gene regions in Z.

20

mays,, which produces epigenetic regulation such as the expression of stress-related genes.15

21

Nickel is an essential mineral for humans; its deficiency can disturb the adsorption of Ca into the

22

bones, which further disturbs Zn metabolism.42 Although the metabolism of Ni in the human

23

body is clearly influenced by Ca and Zn, such mechanisms are not fully understood in plants.

24

Additional studies at the molecular level are needed to explain the mechanisms by which ZnO

25

exposure alters Ni accumulation in subsequent generations. 13



1

Activity of antioxidant enzymes

2

The activity of CAT, APX, and SOD is shown in Figure 4. As evident in Figure 4a and b, none

3

of the treatments significantly affected levels of the stress enzymes APX and CAT relative to the

4

controls. However, seeds from Z-COTE HP1 at 125 mg kg-1 showed increased APX activity

5

relative to seeds from bulk ZnO at the same concentration. Mukherjee et al.43 reported significant

6

increases in CAT and APX activities in leaves of pea plants upon exposure to 125, 250, and 500

7

mg kg-1 of uncoated ZnO NMs.43 Figure 4a shows a slight decrease on APX of S2 seeds from the

8

bulk ZnO at all concentrations, with similar decreases found in roots and leaves of pea plants

9

grown with bulk ZnO at 125, 250, and 500 mg kg-1.43 Figure 4b shows a decreasing trend in

10

CAT in S2 seeds as the concentration of ZnCl2 increases. Negative effects from ZnCl2 were

11

reported in S1 plants23 and seeds.6 Moreover, salt stress reduced CAT activity in Lupinus termis

12

cultivated with 150 mM NaCl.44

13

However, to the best of the authors’ knowledge, there are no reports on the activity of

14

antioxidant enzymes in grains from plants exposed to ZnO NMs. Although the number of studies

15

with coated NMs in plants is limited, it has been reported that in general, surface-modified NMs

16

have different effects in plants than the pristine homologs, often affecting parameters such as

17

metal uptake and accumulation, nutritional quality, root growth, and ROS production.45 For

18

instance, aminopropyltriethoxysilane-coated ZnO NMs increased the biomass of green pea

19

tissues (root, stem and leaf) and the Zn concentration in stems and leaves.8 In S1 seeds from our

20

previous study, Z-COTE HP1 at 125 mg kg-1 was the only compound/concentrations that did not

21

increase Zn accumulation in comparison to the control. The 1.6-fold increase in APX activity of

22

Z-COTE HP1 in comparison to bulk ZnO seeds could be a residual effect somehow related to the

23

different accumulation of Zn in S1 seeds from these treatments.

14


Page 14 of 25

Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Superoxide dismutases are the first line of defense against ROS in the cell.17 As evident in Figure

2

4c, ZnCl2 treatment at 500 mg kg-1 significantly increased in SOD activity S2 compared with the

3

control seeds. Inze and Van Montagu (1995) stated that SOD genes are differentially regulated

4

throughout plant development as a dynamic response to stress conditions. Increases in SOD

5

transcript levels are caused by a rapid turnover of SOD proteins that activates the gene

6

expression of SOD to meet the subsequent cellular demand.46 Although there are no reports on

7

the antioxidant activity in bean seeds exposed to NMs, there is a possibility that the toxic effect

8

15



1 2

Mineral K

3 4 P

5 6 7

S

8 9

Mg

10 11

Fe

12 13 Mn

14 15 16

B

17 18

Mo

19 20

Cu

21 22 Al

23 24 25 26 27 28 29 30

Compound Bulk ZnO

Page 16 of 25

Concentration (mg∙kg dry seeds-1) Cntrl 125 250 500 15772 15913 16173 16817

Z-COTE

15772

16925

16468

15506

Z-COTE HP1

15772

16773

15598

16650

ZnCl2

15772

15539

17353

15834

Bulk ZnO

4813

5090

4975

5222

Z-COTE

4813

5282

5070

4605

Z-COTE HP1

4813

5276

4500

4950

ZnCl2

4813

4423

5690

4554

Bulk ZnO

2325

2323

2391

2459

Z-COTE

2325

2298

2279

2186

Z-COTE HP1

2325

2266

2268

2412

ZnCl2

2325

2249

2434

2162

Bulk ZnO

1812

1861

1849

1996

Z-COTE

1812

1848

1818

1766

Z-COTE HP1

1812

1817

1788

1814

ZnCl2

1812

1805

1942

1779

Bulk ZnO

67.6

65.5

66.1

81.9

Z-COTE

67.6

59.8

63.7

56.0

Z-COTE HP1

67.6

70.7

49.7

68.8

ZnCl2

67.6

62.8

68.8

56.2

Bulk ZnO

15.3

15.3

15.2

16.5

Z-COTE

15.3

14.1

16.6

14.3

Z-COTE HP1

15.3

15.2

13.8

14.5

ZnCl2

15.3

14.9

16.4

13.8

Bulk ZnO

8.9

10.1

10.3

10.6

Z-COTE

8.9

9.1

9.0

9.3

Z-COTE HP1

8.9

8.4

8.6

8.9

ZnCl2

8.9

8.6

9.9

8.7

Bulk ZnO

5.1

5.7

4.6

6.0

Z-COTE

5.1

5.1

5.0

4.7

Z-COTE HP1

5.1

5.4

4.5

4.6

ZnCl2

5.1

4.7

6.8

5.4

Bulk ZnO

4.8

5.3

5.4

6.2

Z-COTE

4.8

5.6

5.2

4.3

Z-COTE HP1

4.8

4.9

4.5

4.7

ZnCl2

4.8

4.1

6.0

3.9

Bulk ZnO

0.09

0.06

0.29

0.05

Z-COTE

0.09

0.44

0.10

0.12

Z-COTE HP1

0.09

0.04

0.00

0.20

ZnCl2

0.09

0.02

0.00

0.00

Figure 2. Mineral elements that remained unaffected in bean seeds cultivated from plants grown in nanoparticle-free soil. Maternal plants were exposed to bulk ZnO, Z-COTE, Z-COTE HP1, and ZnCl2 at 0 (control), 125, 250 and 500 mg kg-1. Values are means of 4 replicates for Zn compounds and 14 for controls. The lighter color represents the lowest accumulation (bottom value) and the darkest color indicates the highest accumulation (top value) for each element. The treatment concentrations refer to S1 plants. 16


Page 17 of 25

Control Control 2500

125125

*

250

500

3

[Ca] Ni (mg · kg dry seeds-1)

Ca (mg · kg dry seeds-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2000 1500 1000 500 0

[Ni]

2.5 2 1.5

*

1

* *

0.5 0

Bulk ZnO

Z-COTE Z-COTE HP1

ZnCl2

Bulk ZnO

Z-COTE Z-COTE HP1

1

Figure 3. Ca and Ni content in bean seeds cultivated from plants grown in nanoparticle-free soil.

2

Maternal plants were exposed to bulk ZnO, Z-COTE, Z-COTE HP1, and ZnCl2 at 0 (control),

3

125, 250 and 500 mg kg-1. Values are mean ± SE of 4 replicates for Zn compounds and 14 for

4

controls. The symbol * represents statistically significant differences between means of the

5

different treatments and the control (p ≤ 0.05). The treatment concentrations refer to S1 plants.

6 7

caused by the Cl- ions in the ZnCl2 exposure (reported in our previous studies)6,23 caused stress

8

in the first generation of plants that activated the gene expression of SOD, which subsequently

9

manifested in the S2 seeds.

17


ZnCl2


Control

125

250

500

(a)

Ascorbate peroxidase

nmol H2O2 (g fresh seed)-1 · min-1

1600

b

1400 1200 1000

nmol H2O2 (g fresh seed)-1 ·min-1

(c)

a

ab

ab

800 600 400 200 0

(b) 600

SOD units (g fresh seed)-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 25

ZnO Bulk

Z-COTE

ZnO Bulk

Z-COTE

Z-COTE HP1

ZnCl2 Catalase

500 400 300 200 100 0 180

Z-COTE HP1 ZnCl2 Superoxidase dismutase

*

160 140 120 100 80 60 40 20 0

Bulk ZnO

Z-COTE Z-COTE HP1 Compound

ZnCl2

1 2 3 4 5 6 7 8 9

Figure 4. Antioxidant activity of (a) ascorbate peroxidase, (b) catalase and, (c) superoxide dismutase in bean seeds cultivated from plants grown in nanoparticle-free soil. Maternal plants were exposed to bulk ZnO, Z-COTE, Z-COTE HP1, and ZnCl2 at 0 (control), 125, 250 and 500 mg kg-1. Values are mean ± SE of 8 replicates for Zn compounds and 32 for controls. Letters represent statistically significant differences between means of the different compounds at the same concentration and the symbol * represents statistically significant differences between means of the different treatments and the control (p ≤ 0.05). The treatment concentrations refer to S1 plants. 18 ACS Paragon Plus Environment

Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Conclusions

2

This study provides evidence that the residual transgenerational effects of parental ZnO NMs

3

exposure were minimal for bean seeds subsequently cultivated in nanoparticle-free soil. Seed

4

production and the time to reach maturity were unaffected. Seeds collected from maternal plants

5

exposed to ZnO NMs, bulk ZnO, and ZnCl2 and yielded S2 seed, retained their nutritional

6

quality, including protein, sugar, and starch content. In addition, levels of the majority of

7

essential elements were unaffected, the exception being Ni and Ca. Nickel was reduced both in

8

the first and second generation of seeds after exposure to ZnO NMs; the highest concentration of

9

bulk ZnO had a residual effect that increased the accumulation of Ca in S2 seeds.

10

The mechanisms for these effects remain unknown, although the potential for contaminant

11

induced genetic or epigenetic changes in the exposed parent is possible and worthy of additional

12

investigation. While the exposure of ZnO NMs did not yield a residual negative effect in

13

antioxidant enzyme activity, the ionic form (ZnCl2) resulted in increased SOD content in S2

14

seeds, also possibly caused by genetic changes upon parental exposure. Further studies at the

15

molecular and genetic level will provide necessary insight into understanding the

16

transgenerational residual impacts of ZnO NMs but in general, this work suggests minimal

17

consequences of the possible incorporation of ZnO NMs into Zn-deficient soils as an agricultural

18

amendment.

19 20

The Supporting Information

21

Antioxidant enzymatic activity in young seeds

19



1 2

Acknowledgements

3

This material is based upon work supported by the National Science Foundation and the

4

Environmental Protection Agency under Cooperative Agreement Number DBI-1266377. Any

5

opinions, findings, and conclusions or recommendations expressed in this material are those of

6

the author(s) and do not necessarily reflect the views of the National Science Foundation or the

7

Environmental Protection Agency. This work has not been subjected to EPA review and no

8

official endorsement should be inferred. Authors also acknowledge the USDA grant 2016-

9

67021-24985 and the NSF Grants EEC-1449500, CHE-0840525 and DBI-1429708. Partial

10

funding was provided by the NSF ERC on Nanotechnology-Enabled Water Treatment (EEC-

11

1449500). This work was also supported by the grant 2G12MD007592 from the National

12

Institutes on Minority Health and Health Disparities (NIMHD), a component of the National

13

Institutes of Health (NIH) and by the grant 1000001931 from the ConTex program. J.L. Gardea-

14

Torresdey acknowledges the Dudley family for the Endowed Research Professorship, the

15

Academy of Applied Science/US Army Research Office, Research and Engineering

16

Apprenticeship program (REAP) at UTEP, grant no. W11NF-10-2-0076, sub-grant 13-7, and the

17

LERR and STARs programs of the University of Texas System. We thank Dr. James Kelly,

18

Michigan State University for kindly supplying bean seeds and John Ranciato from The

19

Connecticut Agricultural Experiment Station for performing the protein quantification. I.A.

20

Medina-Velo acknowledges

21

México.

the Consejo Nacional de Ciencia y Tecnología (CONACYT),

20


Page 20 of 25

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Medina-Velo, I. A.; Adisa, I.; Tamez, C.; Peralta-Videa, J. R.; Gardea-Torresdey, J. L. Effects of Surface Coating on the Bioactivity of Metal-Based Engineered Nanoparticles: Lessons Learned from Higher Plants. In Bioactivity of Engineered Nanoparticles; Yan, B., Zhou, H., Gardea-Torresdey, J., Eds.; Springer: singapore, 2017; pp 43–61.

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Inze, D.; Van Montagu, M. Oxidative Stress in Plants. Curr. Opin. Biotechnol. 1995, 6, 153–158.

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Synopsis

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Bean plants absorb ZnO NMs from soil without transgenerational effects in seeds’ nutrient

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composition, which is of high environmental significance.

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Minimal Transgenerational Effect of ZnO Nanomaterials on the

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